Pleiotropic Role of Rainbow Trout CXCRs in Response to Disease and Environment: Insights from Transcriptional Signatures and Structure Analysis.

Chemokines are cytokines with chemoattractant capacities that exert their physiological functions through the binding of chemokine receptors. Thus, chemokine and receptor complexes exert important roles in regulating development and homeostasis during routine immune surveillance and inflammation. Compared to mammals, the physiology and structure of chemokine receptors in fish have not been systematically studied. Furthermore, the salmonid-specific whole genome duplication has significantly increased the number of functional paralogs of chemokine receptors. In this context, in the current study, trout exhibited 17 cxcr genes, including 12 newly identified and 5 previously identified receptors. Interestingly, gene expression of brain cxcr1 and cxcr4, kidney cxcr3 and cxcr4, and spleen cxcr3, cxcr4, and cxcr5 subtypes were altered by bacterial infection, whereas brain cxcr1, kidney cxcr1 and cxcr7, and liver cxcr2, cxcr3, and cxcr4 subtypes were changed in response to environmental changes. Based on protein structures predicted by ColabFold, the conserved amino acids in binding pockets between trout CXCR4.1 subtypes and human CXCR4 were also analyzed. Our study is valuable from a comparative point of view, providing new insights into the identification and physiology of salmonid chemokine receptors.

Genome-wide detection of positive and balancing signatures of selection shared by four domesticated rainbow trout populations (Oncorhynchus mykiss).

BACKGROUND: Evolutionary processes leave footprints along the genome over time. Highly homozygous regions may correspond to positive selection of favorable alleles, while maintenance of heterozygous regions may be due to balancing selection phenomena. We analyzed data from 176 fish from four disconnected domestic rainbow trout populations that were genotyped using a high-density Axiom Trout genotyping 665K single nucleotide polymorphism array, including 20 from the US and 156 from three French lines. Using methods based on runs of homozygosity and extended haplotype homozygosity, we detected signatures of selection in these four populations. RESULTS: Nine genomic regions that included 253 genes were identified as being under positive selection in all four populations Most were located on chromosome 2 but also on chromosomes 12, 15, 16, and 20. In addition, four heterozygous regions that contain 29 genes that are putatively under balancing selection were also shared by the four populations. These were located on chromosomes 10, 13, and 19. Regardless of the homozygous or heterozygous nature of the regions, in each region, we detected several genes that are highly conserved among vertebrates due to their critical roles in cellular and nuclear organization, embryonic development, or immunity. We identified new candidate genes involved in rainbow trout fitness, as well as 17 genes that were previously identified to be under positive selection, 10 of which in other fishes (auts2, atp1b3, zp4, znf135, igf-1α, brd2, col9a2, mrap2, pbx1, and emilin-3). CONCLUSIONS: Using material from disconnected populations of different origins allowed us to draw a genome-wide map of signatures of positive selection that are shared between these rainbow trout populations, and to identify several regions that are putatively under balancing selection. These results provide a valuable resource for future investigations of the dynamics of genetic diversity and genome evolution during domestication.

Probiotic performance of B. subtilis MS. 45 improves aquaculture of rainbow trout Oncorhynchus mykiss during acute hypoxia stress.

The aim of this study was to produce mutant strains of Bacillus subtilis with high probiotic performance for use in the aquaculture of rainbow trout Oncorhynchus mykiss. The main strain of B. subtilis (MS) was irradiated with gamma rays (5.3 KGy). Subsequently, the B. subtilis mutant strain no. 45 (MS. 45) was selected for bacterial growth performance, resistance to acidic conditions, resistance to bile salts and antibacterial activity against Aeromonas hydrophila and Pseudomonas fluorescens. After 60 days, the rainbow trout (70.25 ± 3.89 g) fed with MS. 45 and MS were exposed to hypoxia stress (dissolved oxygen = 2 ppm). Subsequently, immune indices (lysozyme, bacterial activity and complement activity), hematological indices [hematocrit, hemoglobin, WBC, RBC, mean corpuscular volume (MCV)] and antioxidant factors (T-AOC, SOD and MDA)) were analyzed after and before hypoxia exposure. The expression of immunological genes (IFN-γ, TNF-α, IL-1ß, IL-8) in the intestine and the expression of hypoxia-related genes (HIF-1α, HIF-2α, FIH1) in the liver were compared between the different groups under hypoxia and normoxia conditions. Growth, immunological and antioxidant indices improved in group MS. 45 compared to the other groups. Stress indices and associated immunologic and hypoxia expressions under hypoxia and normoxia conditions improved in MS. 45 compared to the other groups. This resulted in improved growth, immunity and stress responses in fish fed with the microbial supplement of MS. 45 (P < 0.05) under hypoxia and normoxia conditions, (P < 0.05), resulting in a significant improvement in trout aquaculture.

Integrated alternative approaches to select feed-efficient rainbow trout families to enhance the plant protein utilization.

Improving feed utilization efficiency is a challenge in aquaculture. Therefore, we developed an indirect benchmark to use in selecting trout for improved efficiency of feed utilization on plant protein (soy)-based diets, with the long-term goal of reducing the cost of commercial trout production. We used a four-part integrative approach to identify feed efficient individuals among 1595 fish coming from 12 genetically selected families by establishing the phenotypic relationship between feed conversion ratio (FCR) and body weight variations using compensatory feeding regimes. Additionally, we examined the nutritional composition of fish filet for each efficiency phenotype during the compensatory regimen. Our findings showed that the fish with the lowest weight loss during a feed deprivation period and the highest weight gain during the refeeding period (FD-/RF +) demonstrated the lowest FCR (FCR = 0.99) and consisted of individuals from several lines. This finding confirms the possibility of improving feed efficiency in mixed lines. Although feeding period has an effect on nutritional composition of fillet, such selection criteria did not show an effect on groups. Overall, successful selection for the improvement of feed efficiency will have a broad application to commercial fish selective breeding programs, leading to increased aquaculture sustainability in the long run.

Assessing the Effectiveness of Sex-Linked Molecular Markers to Identify Neomale Breeders for the Production of All-Female Progenies of Rainbow Trout.

A cultured stock of masculinized rainbow trout was diagnosed with Y-linked markers (sdY and OmyY1) aiming to detect neomales before their use at the production level. To achieve a reliable diagnosis, the following steps were considered: (1) PCR amplification of the housekeeping ß-actin gene to determine the DNA quality of samples, (2) validation of the Y-linked markers by their PCR amplification in male and female samples with known sex, and (3) molecular sexing of the masculinized juveniles based on male-specific (XY genotype) and neomale-specific (XX genotype) PCR product band patterns visualized on agarose gel. The validity and concordance of the markers were assessed. The housekeeping gene identified samples with negative PCR amplification revealing a poor DNA quality. The OmyY1 marker presented a more distinctive PCR product band pattern between males and females than the sdY marker and identified a higher proportion of true males (sensitivity = 1.0 and 0.91, respectively). The OmyY1 marker accurately identified 105 neomales of the 198 masculinized individuals on account their consistent and distinctive PCR product band pattern. Among both markers, there was a medium high positive concordance (γ index = 0.7). It is concluded that the OmyY1 marker shows the best performance to reliably detect neomales, a step that is essential to have certified breeders for the production of all-female progenies in fish farming.

Chronic cortisol stimulation enhances hypothalamus-specific enrichment of metabolites in the rainbow trout brain.

The hypothalamus is a key integrating center that is involved in the initiation of the corticosteroid stress response, and in regulating nutrient homeostasis. Although cortisol, the principal glucocorticoid in humans and teleosts, plays a central role in feeding regulation, the mechanisms are far from clear. We tested the hypothesis that the metabolic changes to cortisol exposure signal an energy excess in the hypothalamus, leading to feeding suppression during stress in fish. Rainbow trout (Oncorhynchus mykiss) were administered a slow-release cortisol implant for 3 days, and the metabolite profiles in the plasma, hypothalamus, and the rest of the brain were assessed. Also, U-13C-glucose was injected into the hypothalamus by intracerebroventricular (ICV) route, and the metabolic fate of this energy substrate was followed in the brain regions by metabolomics. Chronic cortisol treatment reduced feed intake, and this corresponded with a downregulation of the orexigenic gene agrp, and an upregulation of the anorexigenic gene cart in the hypothalamus. The U-13C-glucose-mediated metabolite profiling indicated an enhancement of glycolytic flux and tricarboxylic acid intermediates in the rest of the brain compared with the hypothalamus. There was no effect of cortisol treatment on the phosphorylation status of AMPK or mechanistic target of rapamycin in the brain, whereas several endogenous metabolites, including leucine, citrate, and lactate were enriched in the hypothalamus, suggesting a tissue-specific metabolic shift in response to cortisol stimulation. Altogether, our results suggest that the hypothalamus-specific enrichment of leucine and the metabolic fate of this amino acid, including the generation of lipid intermediates, contribute to cortisol-mediated feeding suppression in fish.NEW & NOTEWORTHY Elevated cortisol levels during stress suppress feed intake in animals. We tested whether the feed suppression is associated with cortisol-mediated alteration in hypothalamus metabolism. The brain metabolome revealed a hypothalamus-specific metabolite profile suggesting nutrient excess. Specifically, we noted the enrichment of leucine and citrate in the hypothalamus, and the upregulation of pathways involved in leucine metabolism and fatty acid synthesis. This cortisol-mediated energy substrate repartitioning may modulate the feeding/satiety centers leading to the feeding suppression.

Alternative splicing patterns of hnrnp genes in gill tissues of rainbow trout (Oncorhynchus mykiss) during salinity changes.

Alternative splicing (AS) plays an important role in various physiological processes in eukaryotes, such as the stress response. However, patterns of AS events remain largely unexplored during salinity acclimation in fishes. In this study, we conducted AS analysis using RNA-seq datasets to explore splicing patterns in the gill tissues of rainbow trout exposed to altered salinity environments, ranging from 0 ‰ (T0) to 30 ‰ (T30). The results revealed 1441, 351, 483, 1051 and 1049 differentially alternatively spliced (DAS) events in 5 pairwise comparisons, including T6 vs. T0, T12 vs. T0, T18 vs. T0, T24 vs. T0, and T30 vs. T0, respectively. These DAS events were derived from 1290, 328, 444, 963 and 948 genes. Enrichment analysis indicated that these DAS genes were related to RNA splicing and processing. Among these, 14 DAS genes were identified as members of the large heterogeneous nuclear RNP (hnRNP) gene family. Alternative 3' splice site (A3SS), exon skipping (SE) and intron retention (RI) events resulted in the fragmentation or even loss of the functional RNA recognition motif (RRM) domains in hnrnpa0, hnrnp1a, hnrnp1b and hnrnpc genes. The incomplete RRM domains would hinder the interactions between hnRNP genes and pre-mRNAs. It would in turn influence the splicing patterns and mRNA stability of downstream target genes in response to salinity changes. The study provides insights into salinity acclimation in gill tissues of rainbow trout and serves as a significant reference on the osmoregulation mechanisms at post-transcription regulation levels in fish.

The antiviral effects of TRIM23 and TRIM32 proteins in rainbow trout (Oncorhynchus mykiss).

TRIM proteins play a crucial antiviral effector role in the innate immune system of vertebrates. In this study, we found that TRIM proteins exhibited the highest expression levels in immune organs such as spleen and kidney during IHNV infection in rainbow trout, meanwhile, we successfully amplified TRIM23 and TRIM32 from diseased rainbow trout and analyzed their gene sequences, revealing that rainbow trout TRIM23 and TRIM32 proteins are closely related to Atlantic salmon and Chinook salmon; In this experiment, the TRIM23 and TRIM32 protein genes were resoundingly constructed as a recombinant plasmids and expressed in CHSE-214 cells. Upon transfected with the recombinant plasmid, followed by viral infection, significant decreasion in the copy numbers of the virus was observed, indicating that the TRIM23 and TRIM32 proteins of rainbow trout play an important role in inhibiting virus replication, with the TRIM32 role being the most pronounced. These results provide a basis for subsequent in-depth study of the antiviral effects of TRIM proteins, and provide new ideas for immune enhancers.

Genetic diversity patterns in farmed rainbow trout (Oncorhynchus mykiss) populations using genome-wide SNP and haplotype data.

Rainbow trout is one of the most popular aquaculture species worldwide, with a long history of domestication. However, limited information exists about the genetic diversity of farmed rainbow trout populations globally, with most available reports relying on low-throughput genotyping technologies. Notably, no information exists about the genetic diversity status of farmed rainbow trout in Sweden. Double-digest restriction-site-associated DNA sequencing was performed on more than 500 broodfish from two leading producers in Sweden and from the country's national breeding program. Following the detection of single nucleotide polymorphisms (SNPs), genetic diversity was studied by using either individual SNPs (n = 8680; one SNP retained per 300 bp sequence reads) or through SNP haplotypes (n = 20 558; all SNPs retained in 300 bp sequence reads). Similar amounts of genetic diversity were found amongst the three populations when individual SNPs were used. Furthermore, principal component analysis and discriminant analysis of principal components suggested two genetic clusters with the two industry populations grouped together. Genetic differentiation based on the FST fixation index was ~0.01 between the industry populations and ~0.05 when those were compared with the breeding program. Preliminary estimates of effective population size (Ne ) and inbreeding (based on runs of homozygosity; FROH ) were similar amongst the three populations (Ne ≈ 50-80; median FROH ≈ 0.11). Finally, the haplotype-based analysis suggested that animals from the breeding program had higher shared coancestry levels than those from the other two populations. Overall, our study provides novel insights into the genetic diversity and structure of Sweden's three main farmed rainbow trout populations, which could guide their future management.

Involvement of miR-495 in the skin pigmentation of rainbow trout (Oncorhynchus mykiss) through the regulation of mc1r.

MicroRNAs (miRNAs) play crucial roles in skin pigmentation in animals. Rainbow trout (Oncorhynchus mykiss) is a key economic fish species worldwide, and skin color directly affects its economic value. However, the functions of miRNAs in rainbow trout skin pigmentation remain largely unknown. Herein, we overexpressed and silenced miR-495 in vitro and in vivo to investigate its functions. The analysis of spatial and temporal expression patterns suggested that miR-495 is a potential regulator during the process of skin pigmentation. In vitro, mc1r was validated as a direct target for miR-495 by dual-luciferase reporter assay, and overexpression of miR-495 significantly inhibited mc1r expression; in contrast, mc1r and its downstream gene mitf levels were markedly upregulated by decreased miR-495. In vivo, overexpressed miR-495 by injecting agomiR-495 led to a substantial decrease in the expression of mc1r and mitf in dorsal skin and liver, while the opposite results were obtained after miR-495 silencing by antagomiR-495. These findings suggested that miR-495 can target mc1r to regulate rainbow trout skin pigmentation, which provide a potential basis for using miRNAs as target drugs to treat pigmentation disorders and melanoma.

FGF21 Improves Glycolipid Metabolism in Rainbow Trout (Oncorhynchus mykiss) Fed a High-Carbohydrate Diet by Inhibiting Inflammatory Responses and Activating Autophagy.

In this study, the coding region of rainbow trout fgf21 was cloned and sequenced to synthesize a recombinant protein (rFGF21) and investigate its potential role in improving glycolipid metabolism. Acute injection of rFGF21 into rainbow trout effectively reduced serum glucose levels. To investigate the effect of rFGF21 on high-carbohydrate diet (HCD)-induced metabolic disorders in rainbow trout, a 31-day feeding experiment was conducted. At the end of the third week, fish were injected with either PBS or rFGF21. The results showed that the final body weight (FBW) significantly increased in rainbow trout on an HCD (P < 0.05), but there were potential risks including disturbances in glycolipid metabolism and increased inflammatory responses. However, these effects were altered by rFGF21 treatment. In addition, rFGF21 promotes glucose uptake by increasing the phosphorylation levels of AKT (protein kinase B) and GSK3ß (glycogen synthase kinase 3ß), increasing hepatic glycogen, thereby lowering serum glucose. Notably, the rFGF21 did not exacerbate the inflammatory response but downregulated the expression of inflammatory factors. Interestingly, the activation of autophagy and the AMPK pathway may contribute to the positive effect of rFGF21, where rFGF21 injection significantly increased the levels of LC3I/II protein and phosphorylate AMPKα (P < 0.05).

Distinct patterns of inheritance shape life-history traits in steelhead trout.

Life-history variation is the raw material of adaptation, and understanding its genetic and environmental underpinnings is key to designing effective conservation strategies. We used large-scale genetic pedigree reconstruction of anadromous steelhead trout (Oncorhynchus mykiss) from the Russian River, CA, USA, to elucidate sex-specific patterns of life-history traits and their heritability. SNP data from adults returning from sea over a 14-year period were used to identify 13,474 parent-offspring trios. These pedigrees were used to determine age structure, size distributions and family sizes for these fish, as well as to estimate the heritability of two key life-history traits, spawn date and age at maturity (first reproduction). Spawn date was highly heritable (h2 = 0.73) and had a cross-sex genetic correlation near unity. We provide the first estimate of heritability for age at maturity in ocean-going fish from this species and found it to be highly heritable (h2 from 0.29 to 0.62, depending on sex and method), with a much lower genetic correlation across sexes. We also evaluated genotypes at a migration-associated inversion polymorphism and found sex-specific correlations with age at maturity. The significant heritability of these two key reproductive traits in these imperiled fish, and their patterns of inheritance in the two sexes, is consistent with predictions of both natural and sexually antagonistic selection (sexes experience opposing selection pressures). This emphasizes the importance of anthropogenic factors, including hatchery practices and ecosystem modifications, in shaping the fitness of this species, thus providing important guidance for management and conservation efforts.

Cryopreservation effect on DNA methylation profile in rainbow trout spermatozoa.

Spermatozoa are the cells that are most commonly used for cryopreservation of valuable genetic resources in aquaculture. It is known that fish spermatozoa transmit to the embryo not only their genetic but also their epigenetic profile, especially DNA methylation. Therefore, any alteration of the DNA methylation profile in spermatozoa induces the risk of transmitting epigenetic alterations to the offspring. The aim of this study was to assess the effect of cryopreservation on DNA methylation in rainbow trout spermatozoa. To trigger variable cellular response after freezing-thawing, spermatozoa from mature males were cryopreserved with dimethyl sulfoxide, methanol or glycerol as cryoprotectant. We observed that dimethyl sulfoxide was the best to preserve thawed spermatozoa functions. Methanol only slightly preserved all the cellular parameters, while glycerol failed to protect motility and fertilization ability. The consequences on DNA methylation were assessed using Reduced Representation Bisulfite Sequencing (RRBS). Sperm cryopreservation did not thoroughly impact DNA methylation, although 335-564 differentially methylated cytosines were characterized depending on the cryoprotectant. Very few of them were shared between cryoprotectants, and no correlation with the extent of cellular damage was found. Our study showed that DNA methylation was only slightly altered after sperm cryopreservation, and this may render further analysis of the risk for the progeny very challenging.

Evolution of T cell receptor beta loci in salmonids.

T-cell mediated immunity relies on a vast array of antigen specific T cell receptors (TR). Characterizing the structure of TR loci is essential to study the diversity and composition of T cell responses in vertebrate species. The lack of good-quality genome assemblies, and the difficulty to perform a reliably mapping of multiple highly similar TR sequences, have hindered the study of these loci in non-model organisms. High-quality genome assemblies are now available for the two main genera of Salmonids, Salmo and Oncorhynchus. We present here a full description and annotation of the TRB loci located on chromosomes 19 and 25 of rainbow trout (Oncorhynchus mykiss). To get insight about variations of the structure and composition of TRB locus across salmonids, we compared rainbow trout TRB loci with other salmonid species and confirmed that the basic structure of salmonid TRB locus is a double set of two TRBV-D-J-C loci in opposite orientation on two different chromosomes. Our data shed light on the evolution of TRB loci in Salmonids after their whole genome duplication (WGD). We established a coherent nomenclature of salmonid TRB loci based on comprehensive annotation. Our work provides a fundamental basis for monitoring salmonid T cell responses by TRB repertoire sequencing.

Pathological analysis and antimicrobial susceptibility of Chryseobacterium balustinum RTFCP 298 isolated from diseased rainbow trout, Oncorhynchus mykiss.

In this study, six isolates of Chryseobacterium balustinum were characterized from diseased rainbow trout fingerlings. The virulence characteristics, pathogenicity, and antimicrobial susceptibility pattern of these isolates were investigated. The bacterium showed positive results for catalase, cytochrome oxidase, and aesculin hydrolysis, while negative results were obtained for DNase, gelatinase, methyl red, Voges-Proskauer's reaction, Simon citrate, Hydrogen sulphide, and starch hydrolysis. Amino acid metabolism analysis revealed the inability to metabolize arginine, lysine, and ornithine decarboxylase. Molecular characterization (16S rRNA) and phylogenetic analysis revealed the test isolates as C. balustinum, closely related to strain WLT (99.85% similarity) and C. balustinum P-27 (99.77%). Virulence assay indicated haemolytic activity and biofilm formation by the test bacterium. The challenge test confirmed moderate pathogenicity in rainbow trout and established Koch's postulates. The clinical manifestations of infection included fin erosion, eye and body surface haemorrhage, exophthalmia, and organ liquefaction. The minimum inhibitory concentrations of various antimicrobials ranged from 1 to > 256 µg mL-1. The novel synthetic antimicrobial peptides exhibited MICs of 8 to > 256 µg mL-1, suggesting a potential control method. These findings suggest that C. balustinum is an opportunistic pathogen with moderate pathogenicity in rainbow trout. Further research on the host-pathogen relationship is necessary to understand virulence characteristics and pathogenicity in aquaculture.

Potential of low-density genotype imputation for cost-efficient genomic selection for resistance to Flavobacterium columnare in rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Flavobacterium columnare is the pathogen agent of columnaris disease, a major emerging disease that affects rainbow trout aquaculture. Selective breeding using genomic selection has potential to achieve cumulative improvement of the host resistance. However, genomic selection is expensive partly because of the cost of genotyping large numbers of animals using high-density single nucleotide polymorphism (SNP) arrays. The objective of this study was to assess the efficiency of genomic selection for resistance to F. columnare using in silico low-density (LD) panels combined with imputation. After a natural outbreak of columnaris disease, 2874 challenged fish and 469 fish from the parental generation (n = 81 parents) were genotyped with 27,907 SNPs. The efficiency of genomic prediction using LD panels was assessed for 10 panels of different densities, which were created in silico using two sampling methods, random and equally spaced. All LD panels were also imputed to the full 28K HD panel using the parental generation as the reference population, and genomic predictions were re-evaluated. The potential of prioritizing SNPs that are associated with resistance to F. columnare was also tested for the six lower-density panels. RESULTS: The accuracies of both imputation and genomic predictions were similar with random and equally-spaced sampling of SNPs. Using LD panels of at least 3000 SNPs or lower-density panels (as low as 300 SNPs) combined with imputation resulted in accuracies that were comparable to those of the 28K HD panel and were 11% higher than the pedigree-based predictions. CONCLUSIONS: Compared to using the commercial HD panel, LD panels combined with imputation may provide a more affordable approach to genomic prediction of breeding values, which supports a more widespread adoption of genomic selection in aquaculture breeding programmes.

Uniparental and biparental markers unravel invasion pathways, population admixture, and genetic structure in naturalized rainbow trout (Oncorhynchus mykiss).

The present study combined uniparental mtDNA and biparental SNPs to illuminate the invasion and colonization pathways of rainbow trout, Oncorhynchus mykiss, one of the world's most widespread invasive fishes, that has been intensively propagated in Chile, South America. The specific aims of the study were (i) to evaluate potential donor populations, which could be either from the species' native range in North America or from introduced populations in Europe, by comparing mtDNA D-loop/control region haplotypes; and (ii) to assess the factors that have shaped genetic diversity and contemporary genetic structure of rainbow trout populations introduced to Chile through SNP genotyping. The authors comprehensively sampled 24 sites in 12 basins ranging from the High Andean Plateau (Altiplano, 18° S) to northern Patagonia (41° S). Results of the mtDNA data of naturalized trout populations from rivers in the Altiplano (northern Chile) differed from those collected in central and southern Chile, suggesting an origin from North American hatcheries. Naturalized trout populations in central and southern Chile, on the contrary, shared haplotypes with specimens found in European hatcheries. The southern and central Chile populations also contained rare haplotypes, possibly indicating potential spread through aquaculture escapes. Results of the SNP analysis revealed higher allelic richness for trout sampled in sites influenced by commercial aquaculture than sites without commercial aquaculture, likely due to increased admixture between aquaculture broodstock and naturalized trout. The analysis further uncovered some complex patterns of divergent trout populations with low genetic diversity as well as increased relatedness between individuals from isolated sites, suggesting possible local populations. A comprehensive characterization of genetic diversity and structure of rainbow trout should help identify management areas that may augment socioeconomic benefits while preventing the spread and further impacts on biodiversity.

Quantifying impacts of an environmental intervention using environmental DNA.

Environmental laws around the world require some version of an environmental-impact assessment surrounding construction projects and other discrete instances of human development. Information requirements for these assessments vary by jurisdiction, but nearly all require an analysis of the biological elements of ecosystems. Amplicon-sequencing-also called metabarcoding-of environmental DNA (eDNA) has made it possible to sample and amplify the genetic material of many species present in those environments, providing a tractable, powerful, and increasingly common way of doing environmental-impact analysis for development projects. Here, we analyze an 18-month time series of water samples taken before, during, and after two culvert removals in a salmonid-bearing freshwater stream. We also sampled multiple control streams to develop a robust background expectation against which to evaluate the impact of this discrete environmental intervention in the treatment stream. We generate calibrated, quantitative metabarcoding data from amplifying the 12s MiFish mtDNA locus and complementary species-specific quantitative PCR data to yield multispecies estimates of absolute eDNA concentrations across time, creeks, and sampling stations. We then use a linear mixed effects model to reveal patterns of eDNA concentrations over time, and to estimate the effects of the culvert removal on salmonids in the treatment creek. We focus our analysis on four common salmonid species: cutthroat trout (Oncorhynchus clarkii), coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhynchus mykiss), and sockeye salmon (Oncorhynchus nerka). We find that one culvert in the treatment creek seemed to have no impact while the second culvert had a large impact on fish passage. The construction itself seemed to have only transient effects on salmonid species during the two construction events. In the context of billions of dollars of court-mandated road culvert replacements taking place in Washington State, USA, our results suggest that culvert replacement can be conducted with only minimal impact of construction to key species of management concern. Furthermore, eDNA methods can be an effective and efficient approach for monitoring hundreds of culverts to prioritize culverts that are required to be replaced. More broadly, we demonstrate a rigorous, quantitative method for environmental-impact reporting using eDNA that is widely applicable in environments worldwide.

Telomerase Activity in Somatic Tissues and Ovaries of Diploid and Triploid Rainbow Trout (Oncorhynchus mykiss) Females.

Telomerase activity has been found in the somatic tissues of rainbow trout. The enzyme is essential for maintaining telomere length but also assures homeostasis of the fish organs, playing an important role during tissue regeneration. The unique morphological and physiological characteristics of triploid rainbow trout, when compared to diploid specimens, make them a promising model for studies concerning telomerase activity. Thus, in this study, we examined the expression of the Tert gene in various organs of subadult and adult diploid and triploid rainbow trout females. Upregulated Tert mRNA transcription was observed in all the examined somatic tissues sampled from the triploid fish when compared to diploid individuals. Contrastingly, Tert expression in the ovaries was significantly decreased in the triploid specimens. Within the diploids, the highest expression of Tert was observed in the liver and in the ovaries of the subadult individuals. In the triploids, Tert expression was increased in the somatic tissues, while the ovaries exhibited lower activity of telomerase compared to other organs and decreased compared to the ovaries in the diploids. The ovaries of triploid individuals were underdeveloped, consisting of only a few oocytes. The lack of germ cells, which are usually characterized by high Tert expression, might be responsible for the decrease in telomerase activity in the triploid ovaries. The increase in Tert expression in triploid somatic tissues suggests that they require higher telomerase activity to cope with environmental stress and maintain internal homeostasis.

Identification, characterization, and transcription of serotonin receptors in rainbow trout (Oncorhynchus mykiss) in response to bacterial infection and salinity changes.

Serotonergic system is involved in the regulation of physiological functions and behavioral traits including cognition, memory, aggression, stress coping, appetite and immunomodulation. Serotonin exerts its functions via binding distinct serotonin receptors which are classified into 7 groups. Salmonid exhibits expanded functional gene copies due to salmonid-specific whole genome duplication. However, serotonin receptor (htr) repertoire is not fully identified in rainbow trout (Oncorhynchus mykiss). In this study, we identified 39 htr genes, including 14 htr1, 4 htr2, 4 htr2 like, 3 htr3, 4 htr4, 2 htr5, 2 htr6, and 6 htr7 subtypes. We investigated physiological functions of serotonin receptors in response to bacterial pathogens exposure and salinity changes. We showed htr1, htr2, htr4 and htr7 subtypes were associated with immunomodulation in response to Vibrio anguillarum or Aeromonas salmonicida infection. Saltwater (salinity of 15) transfer significantly altered htr1, htr2, htr4, and htr7 subtypes, suggesting trout Htr was associated with osmoregulation. We further showed residues interacted with inverse agonist (methiothepin) and serotonin analogue (5-Carboxamidotryptamine) were conserved between trout and human, suggesting exogenous ligands targeting human HTRs might have a role in aquaculture. This study showed duplicated trout Htrs might be physiologically neofunctionalized and potentially exhibit pleiotropic effects in regulating immunomodulation and osmoregulation.